P185neu-encoding dna and therapeutical uses thereof

ABSTRACT

Plasmids containing sequences encoding different fragments of p185 neu  oncoprotein, able to induce an immune response against tumours expressing oncogenes of the ErbB family, and pharmaceutical compositions thereof.

The present invention relates to plasmid vectors containingp185^(neu)-encoding sequences and the use thereof in DNA vaccinationagainst tumours. The plasmids according to the invention containsequences encoding different fragments of human or rat oncoproteinp185^(neu) and are able to induce a humoral or cell-mediated immuneresponse against tumours expressing oncogenes of the ErbB family.

The invention also relates to pharmaceutical compositions containingsaid plasmids and their use for the prevention or therapy ofp185^(neu)-expressing tumours.

BACKGROUND OF THE INVENTION

Protein p185^(neu), one of the most studied tumour antigens, has raisedgreat interest as target for immune therapy against cancer, due to itspresence on the cell membrane of some of the most common humancarcinomas.

p185^(neu) is a membrane receptor encoded in the rat by proto-oncogeneHer-2/neu and belonging to the family of Class I Tyrosine KinaseReceptors (RTKs), which also comprises the Epidermal Growth FactorReceptor EGF-R (ErbB-1) and other receptors related thereto (ErbB-3,ErbB-4). These receptors are involved in cell proliferation anddifferentiation (Hynes and Stern, 1994 BBA 1198:165) and thereforeattract a great biological and clinical interest. The receptor consistsof three well distinguished domains: an extracellular, transmembrane andintracytoplasmic domain. p185^(neu) is involved in the complex networkof mechanisms of intracellular signal transduction and intracellularcommunication that regulate proliferation and differentiation processes(Boyle 1992 Curr. Op. Oncol. 4:156). Oncogene neu is named after thechemically-induced rat neuroglioblastoma from which it was firstisolated. This activated neu form has a single point mutation thatresults in the replacement of “A” with “T” and in the consequentsubstitution of the Valine residue at position 664 of p185^(neu) with aglutamic acid residue (Val664Glu) (Bargmann et al. 1986, Cell 45:649).

Also the human neu homologous, ErbB-2, has been isolated andcharacterised and it has been demonstrated that both rat HER2/neureceptor and human ErbB2 have a significant homology with EGFR (Coussenset al. 1985, Sciente 230:1132; Yamamoto et al. 1986, Nature 319:230).While a genetic mutation in the rat sequence is the cause ofconstitutive receptor activation through dimerization, in ErbB-2positive human tumours an aberrant expression of the oncogene isobserved (Di Marco et al. 1990, Mol. Cell. Biol. 10: 3247; Klapper etal., 2000, Adv Cancer Res, 77:25), even though, in rare cases,activating point mutations and abnormal splicing mechanisms have beenfound (Kwong et al., 1998, Mol Carcinog, 23:62; Xie et al., 2000, J NatlCancer Inst, 92:412). The overall effect is similar: gene amplificationand increase in the transcription level determine an excess ofp185^(neu) membrane receptor, with consequent increase of active dimersintracellularly transducing growth signals in a ligand-independentmanner.

The crystal structure of human and rat p185^(neu) extracellular regionrecently reported shows that this protein is characterised by a rigidconformation that allows to interact with other ErbB receptors, withoutdirectly binding any ligands, and trigger the proliferation signaltransduction (Cho H S et al. 2003, Nature 421:756).

Under normal circumstances, human p185^(neu) is involved inorganogenesis and epithelial growth; it is expressed at high levelsduring placenta formation and fetal development, whereas it is presentat very low levels in adult tissues (Press et al. 1990, Oncogene 5:953).

Several studies have demonstrated that human p185^(neu) overexpressionis associated to the neoplastic process and to the level of tumoraggression. The overexpression of p185^(neu) has been described in lung(Kern et al. 1986, Cancer Res. 50:5184), colon (Cohen et al. 1989,Oncogene 4:81), ovary (Slamon et al. 1989, Science 244:707)adenocarcinomas and in a high number of human mammary carcinomas (Slamonet al. 1989, Science 244:707; Jardines et al. 1993, Pathobiology61:268).

The fundamental properties that make p185^(neu) an optimal target forplasmid vaccination are: a) its direct involvement in cell growth andcarcinogenesis, therefore clone variants that, due to tumour geneticinstability, lose the expression of this antigen also lose theirtumorigenicity; b) its expression on the plasmatic membrane, which makesit recognizable by antibodies even in tumour cells that lose theexpression of the major histocompatibility system (Lollini P. and FormiG. 2003, Trends Immunol. 24: 62).

Studies carried out on mice transgenic for the activated rat oncogeneHer-2/neu (which spontaneously develop p185^(neu) positive mammarytumours) and on murine models using p185^(neu) positive transplantabletumour lines, have demonstrated the possibility to prevent and curepreneoplastic lesions. As regards in particular the prevention ofmammary tumours in mice transgenic for rat activated Her-2/neu, we havedemonstrated that the plasmid coding for the extracellular andtransmembrane domains of rat p185^(neu) is able to induce an in vivoprotection more effective than the plasmid encoding for the full-lengthrat p185^(neu) or for the extracellular domain only (secreted antigen)(Amici A. et al. 2000, Gene Ther., 7: 703; Rovero S. et al. 2000, J. ofImmunol., 165: 5133). Similar results have been reported by Chen et al.(1998, Cancer Res 58:1965). Other authors have demonstrated thatplasmids encoding for p185^(neu)—either unvaried or mutated so as toeliminate its tyrosine-kinase activity—are effective in preventing theonset of tumours following to p185^(neu)-positive cells inoculum (Wei WZ et al. 1999, Int. J. Cancer 81: 748). Moreover, plasmids devoid of thesignal responsible for the processing through the endoplasmic reticulum(leader), which determines cytoplasmic localization of p185^(neu)antigen, proved equally effective. The protection induced by differentplasmids was mainly mediated by a humoral immune response in the case ofmembrane expression of p185^(neu) and by a T-lymphocyte-mediatedimmune-response in the case of cytoplasmic localization (Pilon S A etal. 2001, J. of Immunol. 167: 3201). However, combined vaccination withplasmids inducing p185^(neu) overexpression in both the cytoplasm andthe membrane was more effective in protecting against tumour growth(Piechocki M P et al. 2001, J. Immunol. 167: 3367).

Thus, the balance between different immune response mechanisms might beparticularly important (Reilly et al., 2001, Cancer Res. 61: 880).Moreover, it has been observed that the vaccination with plasmidsencoding for extracellular and transmembrane domains of rat p185^(neu)is able to eradicate tumour masses with 2 mm diameter, upon inoculum ofcells overexpressing p185^(neu), through a number of different effectormechanisms of the immune system (T helper and T killer cells,antibodies, macrophages, neutrophiles, natural killer cells, Fcreceptors, gamma interferon and perforins), which cooperate to tumorrejection (Curcio C. et al. 2003, J. Clin. Invest. 111: 1161).

DESCRIPTION OF THE INVENTION

Various constructs encoding the human or human/rat chimeric p185 proteinhave been inserted in plasmid vectors and used in immunizationexperiments aimed at preventing tumour progression. For plasmidconstruction, fragments of the human p185^(neu) protein containing thetransmembrane domain and portions of the extracellular domain ofdecreasing length have been prepared from ErbB2 oncogene sequence, orportions thereof have been replaced with homologous sequences from therat Her-2/neu cDNA so as to create chimeric plasmids.

The plasmids thereby obtained have been evaluated in vaccinationexperiments in mice inoculated with tumour cells overexpressing humanp185^(neu). Plasmids containing truncated forms of p185^(neu) induced anantitumor reactivity mediated by killer and helper T lymphocytes, whilechimeric plasmids induced an antibody response against both human andrat p185^(neu).

Based on the results of in vivo experiments, the plasmids containingp185^(neu) sequences able to induce a strong immune response of bothcellular and humoral type have been selected. These plasmids, object ofthe present invention, contain a sequence encoding a p185^(neu) fragmentselected from the group consisting of SEQ ID N. 1-14 (human and ratp185^(neu) reference sequences are available at Gene Bank accessionnumbers M11730 and X03362, respectively).

According to the invention, p185^(neu) encoding sequences can beinserted in any plasmid vectors suitable for human administration.Besides the encoding sequences, the plasmids can contain functionalelements for transcription control, in particular a promoter placedupstream of the encoding sequence, preferably the CMV promoter, startand stop transcription elements, selection markers, such as ampicillinor kanamicin resistance genes, CpG motifs, a polyadenylation site ortranscription activators. Transcription control elements should becompatible with the use of the vector in humans. In a preferredembodiment, the plasmids of the invention contain at least 4 CpG motifs,preferably at least 8, up to a maximum of 80. The CpG motifs(ATAATCGACGTTCAA) (SEQ ID NO: 43) of bacterial origin induce macrophagesto secret IL-12, which in turn induce IFN gamma secretion by naturalkiller cells, thus activating a T helper lymphocyte-mediated response(Chu R. S. et al. 1997, J. Exp. Med., 186: 1623). Therefore, theinsertion of CpG motifs in plasmid sequences enhances the immuneresponse.

In a further embodiment, the invention provides a pharmaceuticalcomposition containing one or more different plasmids as defined abovein association with pharmaceutically acceptable vehicles and excipients.The pharmaceutical compositions, in a form suitable for parenteraladministration, preferably in the form of injectable solution, areconveniently used for DNA vaccination. Principles and methods for DNAvaccination are known to the skilled in the art and are disclosed, forexample, in Liu M A 2003; J Int Med 253: 402.

In another embodiment, the invention provides a combined preparationcontaining at least two, preferably at least four, more preferably atleast eight different plasmids for simultaneous, sequential or separateadministration to a subject or patient.

Plasmids, compositions and preparations according to the invention areused in preventive or therapeutical treatment of subjects at risk ofdeveloping p185^(neu)-positive tumours, or patients with primarytumours, metastasis or relapses of p185^(neu)-positive tumours.Prevention can be primary, when the tumour is not manifest, secondary,when the tumour is in the initial phases as a preneoplastic lesion, otertiary, in the case of tumour relapse or metastatic process. Tumoursthat can benefit from treatment with the plasmids of the invention arethose of epithelial origin, in particular pulmonary, ovary and mammaryadenocarcinomas and, more generally, tumours expressing the p185^(neu)protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of a plasmid containing the sequenceof the second cysteine domain and transmembrane domain of human p185neu.

FIG. 2 illustrates the construction of a chimeric plasmid containing thesequence of the first cysteine domain of rat p185neu and of the secondcysteine domain and transmembrane domain of human (nt 1-1250).

FIGS. 3-9 illustrate the construction of seven plasmids encodingdecreasing fragments of the extracellular domain of human p185neu.

FIGS. 10-14 illustrate the construction of five plasmids encoding fordifferent man-rat chimeric p185neu.

DETAILED DESCRIPTION OF THE INVENTION Construction of the PlasmidBackbone of pCMV3.1

To construct plasmids encoding human p185^(neu) fragments and chimericplasmids, the pCMV3.1 plasmidic backbone was used. Fragments derivingfrom human proto-oncogene ErbB-2 cDNA and from rat proto-oncogeneHer-2/neu cDNA have been inserted in pCMV3.1 (Invitrogen, Milano,Italia) by removing with restriction enzymes DraIII (nt1531) e BsmI(nt3189) a fragment of 1658 bp containing the replication origin f1, thereplication origin and the early SV40 promoter, the gene encoding forneomicine resistance and SV40 polyadenylation signal. The resultingmodified plasmid (pCMV3.1) present some advantages compared to nativepcDNA3.1. In fact, the size reduction to 3900 bp and the removal ofirrelevant sequences contribute to increase transfection efficacy invivo.

Construction of Plasmid pCMV3.1erbB2

Human ErbB2 cDNA, obtained from plasmid pSVerbB2, has been inserted inthe multiple cloning site of pCMV3.1 at restriction sites HindIII andXbaI. This plasmid is used for the construction of plasmids expressingtruncated p185^(neu) and chimeric plasmids.

Construction of Plasmids Containing the Sequence 4XCpG:pCMV3.1hECD-TM-4CpG and pCMV3.1hECD-TM-4-noCpG

After removal of the sequence encoding the intracytoplasmic domain fromplasmid pCMV3.1-erbB2, two plasmids coding for proto-oncogene ErbB2extracellular and transmembrane regions were prepared. The procedurecomprised first the restriction analysis to identify the unique sitespresent in the nucleotide sequence of ErbB2 cDNA. A unique siterecognized by enzyme AccIII (nt 2195) about 20 bp downstream of the endof the transmembrane domain was identified.

The cytoplasmic domain was removed using the enzyme AccIII present asunique restriction site and enzyme XbaI. To re-insert at the 3′ end ofthe DNA of the ErbB2 ECD-TM the nucleotide triplet TAA, recognized astranslation stop signal, we used two synthetic sequences consisting oftwo sense (oligonucleotide #1, #3) and antisense (oligonucleotide #2,#4) oligonucleotides having the restrictions sites AccIII and XbaI attheir ends. In these synthetic sequences there are also four repeatedsequences CpG and noCpG. The latter is used as negative control. Thesetwo new plasmids have been named pCMV3.1hECD-TM-4CpG andpCMV3.1hECD-TM-4-noCpG.

Construction of the Plasmids Containing the Sequence 8XCpG:pCMV3.1H/NhECD-TM-8CpG and pCMV3.1H/NhECD-TM-8noCpG

To add further aspecific immune stimuli we constructed a new plasmidbackbone containing 4 immune-stimulating CpC sequences, called pCMV3.1H/N-4CpG. For this purpose we modified pCMV3.1 so as to remove one ofthe two restriction sites for the enzyme PmeI and invert the restrictionsites for HindIII and NneI present on the multiple cloning site by meansof a synthetic sequence consisting of two sense (oligonucleotide #5) andantisense (oligonucleotide #6) oligonucleotides. In this new plasmid,named pCMV3.1 H/N, two synthetic sequences have been inserted,consisting of two sense (oligonucleotide #7, #9) and antisensenucleotides (oligonucleotide #8, #10), containing four repeats for theCpG and noCpG sequences in the unique restriction sites XbaI and PmeI,thus obtaining pCMV3.1 H/N-4CpG and 4noCpG. Thereafter, DNA fragmentshECD-TM-4CpG and hECD-TM-4-noCpG have been inserted in pCMV3.1H/N-4CpGand in pCMV3.1 H/N-4-noCpG respectively, thus obtaining two new plasmidscalled pCMV3.1H/N-hECD-TM-8CpG and pCMV3.1H/N-hECD-TM-8noCpG.

Construction of the Plasmid Containing the Sequence of the SecondCystein Domain and Transmembrane Domain of Human p185^(neu):pCMV3.1H/Nh2°cysECD-TM-8CpG

Human p185^(neu) extracellular domain is characterised by two regionsrich in cysteins, known as 1^(st) and 2^(nd) cystein sub-domain (1^(st)cys and 2^(nd) cys). Unlike the rat cDNA sequence containing only onesite BstEII (nt1250) in the extracellular domain, located in thenucleotide region that separates 1^(st) cys from 2^(nd) cys, the cDNAsequence of the extracellular domain of ErbB2 has two restriction sitesfor BstEII: in addition to the site in the same position as that of rat(nt1372), a further BstEII site (nt963) is present in the portionencoding the 1^(st) cys of the extracellular domain. Digesting plasmidpCMV3.1H/NhECD-TM-8CpG with HindIII and BstEII, a DNA fragmentconsisting of the 2nd cys from the extracellular domain, thetransmembrane domain, the sequence 8CpG and the plasmid pCMV3.1H/N wasobtained. Then the signal for rat p185neu secretion through theendoplasmic reticulum was inserted by enzymatic DNA amplification (PCRreaction) using a sense oligonucleotide consisting of the primer T7(oligonucleotide #11) which recognizes the T7 RNA polymerase, present atthe beginning of the pCMV3.1H/N multiple cloning site, and an antisenseoligonucleotide (oligonucleotide #12) having the BstEII site at its end.After purification, enzymatic digestion of the amplified fragment withrestriction enzymes HindIII and BstEII and subsequent cloning,pCMV3.1H/Nh2°cys-TM-8CpG (FIG. 1) has been obtained. (FIG. 1). Thisplasmid was used in vaccination experiments, to have it compared withpCMV3.1 H/NhECD-TM-8CpG. Thereafter, a chimeric cDNA encoding for thefusion protein between 2^(nd) cys and transmembrane domain (nt 1372-nt2204) of the human sequence and 1^(st) cys (nt 1-nt 1250) of the ratsequence has been prepared. The reconstitution of the entire proteinsequence by the fusion of portions deriving from rat and human cDNAs,respectively, allows to increase the immune response.

Construction of the Chimeric Plasmid Containing the Sequence of theFirst Cystein Domain of Rat p185neu and of the Second Cystein Domain andTransmembrane Domain of Human (nt 1-nt 1250):pCMV3.1H/N-r1°cys-h2°cysTM-8CpG

Unlike the rat cDNA sequence containing only a BstEII (nt1250) site inthe extracellular domain located in the nucleotide region that separatesthe first and the second region rich in cysteins, the cDNA sequence ofthe extracellular domain of Erb2 has two restriction sites for BstEII:one in position 1372 (nt), as in the rat sequence, and the other inposition 963 (nt), i.e. in the sequence portion encoding for the 1^(st)cys of the extracellular domain. The presence of the BstEII site in thesame position both in the rat cDNA domain (1250 nt) and in the humancDNA (1372 nt) allowed the construction of a plasmid able to encode afusion product between rat 1^(st) cys and human 2^(nd) cys. In fact,digesting pCMV3.1H/N-h2°cysTM-8CpG with restriction enzymes HindIII andBstEII allowed to replace the DNA fragment encoding for rat p185^(neu)secretion signal with the nucleotide sequence encoding for rat 1st cysobtained through digestion of pCMV3.1rECD-TM-4CpG with the same enzymes.The product of plasmid pCMV3.1H/N-r1°cys-h2°cysTM-8CpG (FIG. 2) consistsof a portion of 412 aa of rat p185neu and a portion of 274 aa of humanp185^(neu). This new plasmid, pCMV3.1H/Nr1°cys-h2°cysTM-8CpG has beenused in vaccination experiments using pCMV3.1H/N-hECD-TM-8CpG ascomparative term. Surprisingly, the plasmid coding for the chimericprotein induces in mice a complete protection against tumours expressinghuman p185^(neu) (Table). This protection is similar to that induced bypCMV3.1H/N-hECD-TM-8CpG. Moreover, analysis of the sera of micevaccinated with both plasmids has evidenced a similar antibody titertowards human p185^(neu).

Plasmids Able to Encode Decreasing Fragments of the Extracellular andTransmembrane Domain of Human p185 neu

Construction of seven plasmids that encode decreasing fragments of theextracellular and transmembrane domain of human p185^(neu), namely:pCMV3.1H/NhECD1-TM-8CpG (−70 aa), pCMV3.1H/NhECD2-TM-8CpG (−150 aa),pCMV3.1H/NhECD3-TM-8CpG (−230 aa), pCMV3.1H/NhECD4-TM-8CpG (−310 aa),pCMV3.1H/NhECD5-TM-8CpG (−390 aa), pCMV3.1H/NhECD6-TM-8CpG (−470 aa) andpCMV3.1H/NhECD7-TM-8CpG (−550 aa).

The fragment encoded by the first of these fragments is 70 aa (deletionof 360 bp) shorter. All the others are gradually 80 aa shorter(deletions of 240 bp).

These fragments have been obtained by DNA enzymatic amplification, usingseven different sense oligonucleotides with NheI restriction site(oligonucleotides #13-#19) at its end and an antisense oligonucleotide(oligonucleotide #20) able to recognise the site called “pcDNA3.1/BGHReverse priming site” (830-850 nt) present at the 3′ end of thepolylinker of pCMV3.1. Further to enzymatic digestion with restrictionenzymes NheI and PmeI, amplification products have been cloned inpCMV3.1H/N-neu leader, previously obtained inserting the secretionsignal to the endoplasmic reticulum of rat p185^(neu) in restrictionsites. The DNA fragment of rat p185^(neu) secretion signal has beenobtained by enzymatic DNA amplification using primer T7 (oligonucleotide#11) as sense nucleotide and an antisense nucleotide (oligonucleotide#21) with NheI site at its end. The amplified fragment afterpurification and restriction digestions with HindIII and NheI has beencloned in plasmid pCMV3.1H/N, digested with the same enzymes, thusobtaining the pCMV3.1H/N-neu leader. Membrane expression of thedifferent truncated forms of human p185^(neu) is expected in view of thepresence of the secretion signal to the endoplasmic reticulum of ratp185^(neu). The plasmids encoding the truncated formspCMV3.1H/NhECD1-TM-8CpG (FIG. 3), pCMV3.1H/NhECD2-TM-8CpG (FIG. 4),pCMV3.1H/NhECD3-TM-8CpG (FIG. 5), pCMV3.1H/NhECD4-TM-8CpG (FIG. 6) aswell as the control plasmid pCMV3.1H/NhECD-TM-8CpG, protect 100% of thevaccined mice against a lethal inoculum of tumour cells expressing humanp185^(neu) (Table). Plasmid pCMV3.1H/NhECD5-TM-8CpG (FIG. 7) protects60% of the animals (Table), while plasmids pCMV3.1H/NhECD6-TM-8CpG andpCMV3.1H/NhECD7-TM-8CpG (FIG. 8, 9), do not have protective effectagainst a lethal inoculum of tumour cells expressing human p185^(neu)(Table). The protein products expressed by the different plasmids arenot secreted through the endoplasmic reticulum. The absence of consensussequences necessary for glycosilation and for their processing throughthe endoplasmic reticulum, or conformational changes due to deletion ofaminoacids at the —NH₂ terminus, could explain the absence of proteinproducts in the membrane. Therefore, to further verify if the varioustruncated forms of the extracellular and transmembrane domain of humanp185^(neu) were correctly expressed, new plasmids coding for fusionproteins characterized by epitope myc at the —NH₂ terminus weregenerated. These recombinant proteins are recognized by an anti-mycmonoclonal antibody, therefore it is possible to analyse theirexpression and localisation by confocal microscopy.

First a new plasmid coding for the secretion signal to the ratendoplasmic reticulum (neu leader) and for the myc epitope has beencreated. Cloning has been carried out using a synthetic sequenceconsisting of a sense (oligonucleotide #22) and antisense(oligonucleotide #23) having at both ends the NheI site. The NheI sitein the 5′ position was mutated so that, once correctly ligated, it wasnot recognized by the enzyme. We thus obtained thepCMV3.1H/Nneuleader-myc epitope. With this plasmid, the sequencesencoding human p185^(neu) truncated forms have been cloned in therestriction sites NheI and PmeI. Then, 3T3 NIH fibroblasts have beentransfected in vitro with plasmids using lipofectamine 2000 (Invitrogen,Milan, Italy). After 48 hours the transfected cells have been analysedwith confocal microscopy, using a FITC-conjugated anti-myc monoclonalantibody (Sigma-Aldrich Srl, Milan, Italy). It has been thusdemonstrated that all the plasmids-encoded truncated forms are locatedin the cytoplasm. 3T3 NIH fibroblasts have been transfected in parallelwith plasmid pCMV3.1H/NhECD-TM-8CpG and analysed with confocalmicroscopy using c-erbB2/c-neu Ab-3 monoclonal antibody (Oncogene,Boston, Mass.) as primary antibody and a FITC-conjugated anti-mousesecondary antibody (PharMigen, San Diego, Calif.). It was thus observedthat human ECD-TM is expressed in the membrane. The results obtainedusing the first four plasmids described previously(pCMV3.1H/NhECD1-TM-8CpG, pCMV3.1H/NhECD2-TM-8CpG,pCMV3.1H/NhECD3-TM-8CpG, pCMV3.1H/NhECD4-TM-8CpG), demonstrate that acellular response is sufficient for antitumour prevention. However, itis known that contemporaneous activation of the cellular and humoralresponse is necessary for a more effective therapy (Rielly et al., 2001,Cancer Res 61:880). As already described in the previous paragraph, thechimeric protein encoded by plasmid pCMV3.1H/N-r1°cys-h2°cysTM-8CpG isable to protect 100% of the vaccined animals and is able to induce astrong humoral response in the mice.

Chimeric Plasmids Able to Encode for Five Different Man-Rat Chimericp185^(neu)

For the construction of plasmids coding for chimeric proteins, weselected pCMV3.1H/NhECD1-TM-8CpG, pCMV3.1H/NhECD2-TM-8CpG,pCMV3.1H/NhECD3-TM-8CpG and pCMV3.1H/NhECD4-TM-8CpG. These four plasmidsprotect 100% of the vaccinated mice against a lethal inoculum of tumourcells expressing human p185^(neu). Also plasmid pCMV3.1H/NhECD5-TM-8CpGhas been selected, even if it protects only 60% of the vaccinated mice,because the encoded protein differs only by 17 aa from that encoded bypCMV3.1H/Nh2°cysECD-TM-8CpG (275 aa), which protects 20% of thevaccinated mice. We can hypothesize that the peptide sequence of 17 aacorresponds to an important epitope for the induction of an effectiveimmune response.

DNA fragments encoding for rat p185^(neu) portions have been obtained byDNA enzymatic amplification. To amplify these cDNA fragments sixoligonucleotides having all the same orientation, namely that of T7primer (oligonucleotide #11), have been used, while the five antisensehave been designed to recognize rat cDNA in the proper positions andhave the restriction site for NheI at their ends (oligonucleotides#24-#28). After purification and digestion with restriction enzymesHindIII and NheI, the amplified fragments have been inserted in thecorresponding plasmids (pCMV3.1H/NhECD1-TM-8CpG,pCMV3.1H/NhECD2-TM-8CpG, pCMV3.1H/NhECD3-TM-8CpG,pCMV3.1H/NhECD4-TM-8CpG pCMV3.1H/NhECD5-TM-8CpG) and digested with thesame restriction enzymes. In this way we obtained five new plasmids ableto code for chimeric proteins of 689 aa, of which 2 (Val-Ser) belong torestriction site NheI used for the conjunction between rat and humanDNA. The presence of these two aa renders both human and rat portionsheteroclytic.

The chimeric proteins differ for human p185^(neu) decreasing portionsand rat p185^(neu) increasing portions. PlasmidpCMV3.1H/Nr73-hECD1-TM-8CpG (FIG. 10) encodes 73 aa of the ratp185^(neu) extracellular domain and 614 aa of human p185^(neu). PlasmidpCMV3.1H/Nr153-hECD2-TM-8CpG (FIG. 11) encodes 153 aa of the ratp185^(neu) extracellular domain and 534 aa of human p185^(neu). PlasmidpCMV3.1H/Nr233-hECD3-TM-8CpG (FIG. 12) encodes 233 aa of the ratp185^(neu) extracellular domain and 454 aa of human p185^(neu). PlasmidpCMV3.1H/Nr313-hECD4-TM-8CpG (FIG. 13) encodes 313 aa of the ratp185^(neu) extracellular domain and 374 aa of human p185^(neu). PlasmidpCMV3.1H/Nr393-hECD5-TM-8CpG (FIG. 14) encodes 393 aa of the ratp185^(neu) extracellular domain and 294 aa of human p185^(neu). Indirectevidence of the membrane expression of human/rat chimeric p185^(neu)encoded by these plasmids has been obtained immunizing mice with thefive new plasmids and with pCMV3.1H/N-r1°cys-h2°cysTM-8CpG as positivecontrol. The sera of all vaccinated mice contain specific antibodiesagainst human p185^(neu). Moreover, the animals vaccinated with plasmidsencoding the five different chimeric proteins are also protected with alethal inoculum of tumour cells expressing human p185^(neu).

EXAMPLES Example 1 Construction of PlasmidpCMV3.1H/N-r1°cys-h2°cysTM-8CpG

To construct chimeric plasmid pCMV3.1H/N-r1°cys-h2°cysTM-8CpG we startedfrom plasmid pCMV-ECD-TM, which expresses the extracellular andtransmembrane domain of rat p185^(neu) (Amici et al 2000, Gene Ther., 7:703). pCMV-ECD-TM was digested with restriction enzymes HindIII and XbaI(BioLabs, Beverly, Mass.) to separate the insert from the plasmidbackbone.

Restriction Digestion with EnzymeHindIII:

plasmid DNA (1 μg/μl) 10 μl restriction buffer 10X (NEB 2) 10 μl HindIII(10 U/μl) 5 μl H₂O 75 μl 100 μl final volume

The mixture was incubated at 37° C. for 4 hours and the digestionproduct controlled by electrophoresis on 1% agarose gel using amolecular weight marker and undigested plasmid as control.

Once confirmed plasmid linearization, DNA was precipitated by adding1/10 volume of 3 M sodium acetate at pH 5.2 and 2 volumes of coldabsolute ethanol.

The sample was kept on ice for 20 min., then centrifugated with aminicentrifuge at 14.000 rpm for 12 min. The pellet was washed threetimes with 1 ml 70% cold ethanol, dried under vacuum for 5 min, thenresuspended in 84 μl H₂O and enzymatically digested with restrictionenzyme XbaI.

Restriction Digestion with Enzyme XbaI:

DNA resuspended in H₂O (10 μg) 84 μl Restriction buffer 10X (NEB2) 10 μlBSA 100X (100 mg/ml) 1 μl XbaI (10 U/ml) 5 μl 100 μl

The mixture was incubated at 37° C. for 4 hours and the digestionproduct was precipitated and dried as described above. DNA wasresuspended in 30 μl H₂O.

The two DNA fragments corresponding to the plasmid backbone (pCMV of4400 bp) and to the insert (ECD-TM of 2100 bp) were separated byelectrophoresis on a 1% agarose gel.

The band corresponding to the insert was removed and DNA eluted from thegel using a Qiaquick gel extraction kit (Qiagen Italy).

In parallel, the new plasmid backbone (pCMV3.1H/N-4CpG) wherein the DNAfragment corresponding to rat p185 ECD-TM, was digested with the samerestriction enzymes and eluted on agarose gel.

The DNA fragments corresponding to rat ECD-TM and the linearized plasmidpCMV3.1H/N-4CpG were used to obtain pCMV3.1H/N-rECD-TM-4CpG by ligationreaction.

Ligation Reaction

DNA insert (rECD-TM) (50 ng/μl) 2 μl Linearized plasmid DNA(pCMV3.1H/N4CpG) (50 ng/μl) 1 μl Reaction buffer 10X for T4 DNA ligase 1μl T4 DNA ligase (2 U/μl) 1 μl H₂O 5 μl 10 μl 

The ligation reaction was incubated at 16° C. for 4 hours.

The ligation product was then used to transform the E. coli bacterialstrain DH5α. The bacterial cells have been made competent with the CaCl₂technique.

Transformation of the bacterial strain DH5α:

Competent bacterial cells 100 μl Ligation product  5 μl

To make the plasmid DNA penetrate the competent cells, these were kepton ice for 40 min. and submitted to thermal shock (1.5 min. at 42° C.and then 2 min. on ice).

After adding 1 ml LB growth medium, the transformed bacterial cells wereincubated at 37° C. for 1 hour to restore their physiologicalconditions.

The cell suspension was then centrifuged at 6000 rpm for 1 min. and thepellet was resuspended in 100 μl LB.

The cells were seeded in Petri dishes containing selective solid medium(LB with agar+ampicillin 100 μg/ml) and grown at 37° C. for 1 night.Ampicillin allows the growth of cells containing plasmidpCMV3.1H/N-rECD-TM-4CpG which confers ampicillin-resistance.

The resulting clones were analysed by alkaline lysis to select thosecontaining the recombinant plasmid pCMV3.1H/N-rECD-TM-4CpG.

To obtain chimeric plasmid pCMV3.1H/N-r1°cys-h2°cysTM-8CpG, plasmidpCMV3.1H/N-rECD-TM-4CpG was digested with restriction enzymes BstEII andXbaI to remove the second cystein domain together with the transmembranedomain of rat p185^(neu). At the same time, plasmid pCMV3.1hECD-TM-4CpGwas digested with the same enzymes to isolate the DNA fragmentcorresponding to the second cystein subdomain and transmembrane domainof the human gene.

Digestion with BstE11:

plasmid DNA (1 μg/μl) 10 μl Restriction buffer 10X (NEB3) 10 μlBstEII(10 U/μl) 5 μl H₂O 75 μl 100 μl final volume

The mixture was incubated at 60° C. for 4 hours.

Restriction digestion with XbaI, recovery of the fragments to be usedfor cloning, ligation reaction and transformation of competent cellshave been described previously.

The resulting chimeric plasmid pCMV3.1H/N-r1°cys-h2°cysTM-8CpG has beensequenced using the automatic ABI PRISM 310 Genetic Analyzer (AppliedBiosystem), to verify the correct insertion of the fragmentcorresponding to the 2^(nd) cystein subdomain and the transmembranedomain of the human gene.

Example 2 In Vivo Test

Animals

Balb/cAnCr (H-2^(d)) female mice aged about seven weeks have been usedfor all experiments.

The animals, supplied by Charles River Laboratories (Calco, MI, Italy),are grown in aseptic conditions and in accordance with the EuropeanCommunity guidelines.

Intramuscular Administration Followed by In Vivo Electroporation

To avoid unwanted contractions of the tibial muscle each mouse wasanaesthetized by i.p. inoculum of 300 μl avertine, made of 0.58 gtribromoethanol (Sigma-Aldrich) and 310 μl Tert-Amyl alcohol (Aldrich)dissolved in 39.5 ml deionized H₂O. All mice have been then shaved incorrespondence of the tibial muscle for the inoculum.

The animals have been vaccinated in correspondence of both antero-tibialmuscles, with 40 μl of solution containing 50 μg DNA.

The DNA-containing mixture was prepared shortly before use, inconformity with the indications of Dr. F. Pericle (Valentis, Inc., TheWoodlands, Tex., USA). This solution contains 1.25 mg/ml plasmid DNA, 6mg/ml poly-L-glutamate sodium salt (Sigma-Aldrich, S.r.l., Milano,Italia), 150 mM sodium chloride (Fluka, BioChemika, Buchs, Switzerland)and distilled water free from endotoxins (Nucleare Free Water, PromegaCorporation) to a final volume of 1 ml.

After about 5 min from the inoculum, the treated area was submitted toelectroporation, by application of two electric impulses having anintensity of 375 V/cm², each lasting 25 ms, using the electroporatorElectro Square Porator (T820, BTX, San Diego, Calif., USA). Thetranscutaneous electric impulses have been applied by use of two squaresteel electrodes placed at mm from each other, beside each paw. Geneimmunization by electroporation was carried out twice for each animal 21and 7 days before inoculum of tumour cells.

Inoculum of Tumour Cells

The mice have been inoculated with a suspension containing 2×10⁵ D2F2/E2cells. These cells derive from a mammary tumour spontaneously generatedin a hyperplastic alveolar node of a BALB/c mouse and express highlevels of human p185.

In Vivo Evaluation of Tumour Growth

Tumour growth was evaluated weekly by palpation and the dimensions ofthe tumours were measured along two perpendicular diameters with acalibre. Neoplastic masses measuring more than 3 mm are considered astumours.

Tumour growth was followed for 100 days from tumour inoculum or untilthe tumour had grown to a diameter higher than 10 mm, then animals weresacrificed.

TABLE Mice: female BALB/c Tumour: D2F2-E2 espressing human p185^(neu)plasmids Number of mice protection antibodies pCMV3.1H/N-8CpG 5  0% −pCMV3.1H/N-hECD- 5 100% +++ TM-8CpG pCMV3.1H/N-hECD1- 5 100% − TM-8CpGpCMV3.1H/N- hECD2- 5 100% − TM-8CpG pCMV3.1H/N- hECD3- 5 100% + TM-8CpGpCMV3.1H/N- hECD4- 5 100% ++ TM-8CpG pCMV3.1H/N- hECD5- 5  60% − TM-8CpGpCMV3.1H/N- hECD6- 5  0% − TM-8CpG pCMV3.1H/N- hECD7- 5  0% − TM-8CpGpCMV3.1H/N-r1°cys- 5 100% +++ h2°cys.-TM-8CpG

List of Oligonucleotides Synthesized and Used for Plasmid Construction

#1. AccIII-TAA-4CpG-erbB2 sense 71 nt (SEQ ID NO: 15)5′CCGGAAGTAAATAATCGACGTTCAAATAATCGACGTTCAAATAATCGACGTTCAAATAATCGACGTTCAAT 3′ #2. XbaI-TAA-4CpG-erbB2 sense 71 nt(SEQ ID NO: 16) 5′CTAGATTGAACGTCGATTATTTGAACGTCGATTATTTGAACGTCGATTATTTGAACGTCGATTATTTACTT 3′ #3. AccIII-TAA-4CpG-erbB2 sense 71 nt(SEQ ID NO: 17) 5′CCGGAAGTAAATAATAGAGCTTCAAATAATAGAGCTTCAAATAATAGAGCTTCAAATAATAGAGCTTCAAT 3′ #4. XbaI-TAA-4CpG-erbB2 sense 71 nt(SEQ ID NO: 18) 5′CTAGATTGAAGCTCTATTATTTGAAGCTCTATTATTTGAAGCTCTATTATTTGAAGCTCTATTATTTACTT 3′ #5. HindIII-NheI sense 27 nt(SEQ ID NO: 19) 5′ CTAGGAAGCTTGTTTAACTTGCTAGCT 3′#6. HindIII-NheI antisense 27 nt (SEQ ID NO: 20)5′AGCTAGCTAGCAAGTTAAACAAGCTTC 3′ #7. XbaI-4CpG-neu sense 68 nt(SEQ ID NO: 21) 5′CTAGATAATCGACGTTCAAATAATCGACGTTCAAATAATCGACGTTCAAATAATCGACGTTCAAGTTT 3′ #8. PmeI-CpG-neu antisense 64 nt(SEQ ID NO: 22) 5′AAACTTGAACGTCGATTATTTGAACGTCGATTATTTGAACGTCGATTATTTGAACGTCGATTAT 3′ #9. XbaI-4noCpG-neu sense 68 nt(SEQ ID NO: 23) 5′CTAGATAATAGAGCTTCAAATAATAGAGCTTCAAATAATAGAGCTTCAAATAATAGAGCTTCAAGTTT 3′ #10. PmeI-4noCpG-neu-antisense 64 nt(SEQ ID NO: 24) 5′AAACTTGAAGCTCTATTATTTGAAGCTCTATTATTTGAAGCTCTATTATTTGAAGCTCTATTAT 3′ #11. T7 primer (SEQ ID NO: 25)5′TAATACGACTCACTATAGGG 3′ #12. BstEII-neuleader antisense 32 nt(SEQ ID NO: 26) 5′GGCCGGTTACCCGCGATTCCGGGGGGCAGGAG 3′#13. hECD1-TM-sense-NheI 35 nt (SEQ ID NO: 27)5′CCGGCTAGCTAGCCTGTCCTTCCTGCAGGATATCC 3′ #14. hECD2-TM-sense-NheI 35 nt(SEQ ID NO: 28) 5′CCGGCTAGCTAGCGGAGGGGTCTTGATCCAGCGGA 3′#15. hECD3-TM-sense-NheI 35 nt (SEQ ID NO: 29)5′CCGGCTAGCTAGCCTGCCCACTGACTGCTGCCATG 3′ #16. hECD4-TM-sense-NheI 35 nt(SEQ ID NO: 30) 5′CCGGCTAGCTAGCTGCACCCTCGTCTGCCCCCTGC 3′#17. hECD5-TM-sense-NheI 35 nt (SEQ ID NO: 31)5′CCGGCTAGCTAGCCCGCTCCAGCCAGAGCAGCTCC 3′ #18. hECD6-TM-sense-NheI 35 nt(SEQ ID NO: 32) 5′CCGGCTAGCTAGCAACACCCACCTCTGCTTCGTGC 3′#19. hECD7-TM-sense-NheI 35 nt (SEQ ID NO: 33)CCGGCTAGCTAGCCCCAGGGAGTATGTGAATGCCA 3′#20. pcDNA3.1/BGH Reverse primer 20 nt (SEQ ID NO: 34)5′TAGAAGGCACAGTCGAGGCT 3′ #21. NheI-neuleader-antisense 43 nt(SEQ ID NO: 35) 5′CCGGCTAGCTAGCCGCGATTCCGGGGGGCAGGAGGGCGAGG AG 3′#22. His-myc-sense-noNheI 69 nt (SEQ ID NO: 36)5′CTAGGCATCATCATCATCATCATAATGGTCATACCGGTGAACAAAAACTCATCTCAGAAGAGGATCTGG 3′ #23. His-myc-antisense-NheI 69 nt(SEQ ID NO: 37) 5′CTAGCCAGATCCTCTTCTGAGATGAGTTTTTGTTCACCGGTATGACCATTATGATGATGATGATGATGC 3′ #24. NheI-73neu antisense 35 nt(SEQ ID NO: 38) 5′CCGGCTAGCTAGCGCTGGCATTGGCAGGCACGTAG 3′#25. NheI-153neu antisense 35 nt (SEQ ID NO: 39)5′CCGGCTAGCTAGCCAGGATCTCTGTGAGACTTCGA 3′#26. NheI-233neu antisense 35 nt (SEQ ID NO: 40)5′CCGGCTAGCTAGCGCCCTTGCACCGGGCACAACCA 3′ #27.  (SEQ ID NO: 41)5′CCGGCTAGCTAGCTCCCACTTCCGTAGACAGGTAG 3′#28. NheI-393neu antisense 35 nt (SEQ ID NO: 42)5′CCGGCTAGCTAGCAATGCCGGAGGAGGGGTCCCCA 3′

1. A DNA transfer vector comprising at least one of the nucleotidesequences selected from the group consisting of SEQ ID NO: 1, 3, 4, 5,6, 8, 9, 10 and
 11. 2. The DNA transfer vector according to claim 1,wherein said vector is a plasmid.
 3. The DNA transfer vector accordingto claim 2, further comprising a transcription promoter.
 4. The DNAtransfer vector according to claim 3, wherein the transcription promoteris CMV.
 5. The DNA transfer vector according to claim 2, furthercomprising 4 CpG motifs.
 6. The DNA transfer vector according to claim5, comprising 8 CpG motifs.
 7. A pharmaceutical composition comprising aDNA transfer vector according to claim 1 in admixture with apharmaceutically acceptable vehicle and excipient.
 8. The pharmaceuticalcomposition according to claim 7, wherein said composition is in a formfor parenteral administration.
 9. A pharmaceutical compositioncomprising at least two different DNA transfer vectors according toclaim 1 in admixture with a pharmaceutically acceptable vehicle andexcipient.
 10. The DNA transfer vector according to claim 1, comprisingthe nucleotide sequence of SEQ ID NO:
 1. 11. The DNA transfer vectoraccording to claim 1, comprising the nucleotide sequence of SEQ ID NO:3.
 12. The DNA transfer vector according to claim 1, comprising thenucleotide sequence of SEQ ID NO:
 4. 13. The DNA transfer vectoraccording to claim 1, comprising the nucleotide sequence of SEQ ID NO:5.
 14. The DNA transfer vector according to claim 1, comprising thenucleotide sequence of SEQ ID NO:
 6. 15. The DNA transfer vectoraccording to claim 1, comprising the nucleotide sequence of SEQ ID NO:8.
 16. The DNA transfer vector according to claim 1, comprising thenucleotide sequence of SEQ ID NO:
 9. 17. The DNA transfer vectoraccording to claim 1, comprising the nucleotide sequence of SEQ ID NO:10.
 18. The DNA transfer vector according to claim 1, comprising thenucleotide sequence of SEQ ID NO: 11.